Methods for manipulating yield of plants and identifying yield genes

ABSTRACT

Methods for manipulating yield and generation time of plants, especially short day plants such as soybean are provided. The methods comprise manipulating external signals such as long day conditions, short day conditions, growth medium, and nutrient supply.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 61/095,126, filed Sep. 8, 2008, which is hereby incorporated byreference herein in its entirety.

FIELD

The present invention discloses methods for manipulating yield of plantsand identifying yield genes. More specifically, the present inventiondiscloses methods for manipulating seed yield of short day plants suchas soybean plants and identifying yield genes from soybean plants.

BACKGROUND

Plant growers are always looking for new methods for manipulating yieldof a plant, especially for enhancing seed yield of an economicallyimportant plant such as soybean. Further, manipulation of seed yield orseed production time is useful in advancing a seed quickly through theproduct development phases such as through growth chamber, nursery,green house, and field testing.

Applicants have been able to manipulate the yield of a short day plantsuch as soybean plant by manipulating its vegetative and floweringresponses with external signals such as exposure to long day growingconditions, exposure to short day growing conditions, growth medium, andnutrient supply. Further, the method can be used for identifying yieldgenes involved in such as genes involved in early induction offlowering, pod set, and retention and/or abscission of flowers and pods.

SUMMARY

A method for manipulating yield of a short day plant is provided. Themethod includes a step of initiating growth of at least one short dayplant under long day growing conditions. The short day plant is selectedfrom the group consisting of soybean, cotton, rice, sugarcane, tobacco,and strawberry. The long day growing conditions comprise at least about14 hours of light per day at a light intensity of from about 1000 toabout 2000 μmoles m⁻¹s⁻¹ and a temperature of from about 84° F. to about90° F. and a night temperature of from about 62° F. to about 70° F.

The method further comprises controlling the environment of the shortday plant to provide for short day growing conditions for about 3 toabout 21 days. The period of short day growing conditions is initiatedat a plant growth stage of from about V1 to about V4. The short daygrowing conditions comprise maintaining about 9 to about 11 hours oflight per day at light intensity of about 700 to about 900 μmoles μmolesm⁻¹s⁻¹ and a temperature of from about 78° F. to about 82° F. and about14 hours of night at a temperature of from about 66° F. to about 70° F.

The method further comprises returning the plant to long day growingconditions. As described above, the long day growing conditions compriseat least about 14 hours of light per day at a light intensity of fromabout 1000 to about 2000 μmoles m⁻¹s⁻¹ and a temperature of from about84° F. to about 90° F. and a night temperature of from about 62° F. toabout 70° F.

In another embodiment, the method of the present invention furthercomprises growing the short day plant under conditions that restrictsvegetative growth and enhances flowering. Such conditions comprisegrowing the short day plant in a soil volume of about 2.0 mL to about4.0 mL per seed to be produced.

In another embodiment, the method of the present invention furthercomprises providing the short day plant nutrients sufficient to supportseed development. Such nutrients may be selected from the groupconsisting of Calcium Nitrate, Phosphate, micronutrients, and MagnesiumSulfate wherein the amount of nutrients supplied provides a soil EC offrom about 1.0 to about 1.6 mmhos and a soil pH of from about 5.1 toabout 6.0.

A method for manipulating yield of a soybean plant is also provided. Themethod comprises initiating growth of at least one soybean plant underlong day growing conditions. The long day growing conditions comprise atleast about 14 hours of light per day at a light intensity of about 1000to about 2000 μmoles m⁻¹ s⁻¹ and a temperature of from about 84° F. to90° F. and a night temperature of from about 62° F. to about 70° F.

The method further comprises controlling the environment of the soybeanplant to provide for short day growing conditions for about 3 to about21 days. The period of short day growing conditions is initiated at aplant growth stage of from about V1 to about V4. The short day growingconditions comprise maintaining about 9 to about 11 hours of light perday at a light intensity of from about 700 to about 900 μmoles m−1s−1and a temperature of from about 78° F. to about 82° F. and about 14hours of night at a temperature of from about 66° F. to about 70° F.

The method further comprises returning the plant to long day growingconditions. As described above, the long day growing conditions compriseat least about 14 hours of light per day at a light intensity of fromabout 1000 to about 2000 μmoles m⁻¹s⁻¹ and a temperature of from about84° F. to 90° F. and a night temperature of from about 62° F. to about70° F.

In another embodiment, the method of the present invention furthercomprises growing the soybean plant under conditions that restrictsvegetative growth and enhances flowering. Such conditions comprisegrowing the soybean plant in a soil volume of from about 2.0 mL to about4.0 mL for every seed to be produced.

In yet another embodiment, the method of the present invention furthercomprises providing the soybean plant nutrients sufficient to supportseed development. Such nutrients may be selected from the groupconsisting of Calcium Nitrate, Phosphate, micronutrients, and MagnesiumSulfate wherein the amount of nutrients supplied provides a soil EC offrom about 1.0 to about 1.6 mmhos and a soil pH of from about 5.1 toabout 6.0.

Still further, another embodiment of the invention comprises methods foridentifying genes conferring increased yield in a short day plant. Themethods generally comprise initiating growth of at least one short dayplant under long day growing conditions and controlling the environmentof the at least one short day plant to provide for short day growingconditions for about 3 to about 21 days. The at least one short dayplant is then returned to long day growing conditions and tissue isharvested from the at least one short day plant. Transcriptionalprofiling is then performed on the harvested tissue to identifypotential genes that may confer increased yield in the at least oneshort day plant.

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Generally, the nomenclatureused herein and the manufacture or laboratory procedures described beloware well known and commonly employed in the art. Conventional methodsare used for these procedures, such as those provided in the art andvarious general references. Where a term is provided in the singular,the inventors also contemplate aspects of the invention described by theplural of that term. Where there are discrepancies in terms anddefinitions used in references that are incorporated by reference, theterms used in this application shall have the definitions given herein.The inventors do not intend to be limited to a mechanism or mode ofaction. Reference thereto is provided for illustrative purposes only.

The present description discloses methods for manipulating seed yieldper plant. In one embodiment, it is the seed yield of a short day plant.In another embodiment, it is the seed yield of a soybean plant. Thepresent description also discloses methods for reducing the seedgeneration time of a plant. In one embodiment, it is the seed generationtime of a short day plant. In another embodiment, it is the seedgeneration time of a soybean plant.

The methods described herein comprise the manipulation of externalvegetative and reproductive signals to control seed production. Forexample, by using the instant methods, a soybean plant can bemanipulated to produce a specific amount of seeds in a required time fora specific project. For example, as described in Table 2 below, asoybean plant can be manipulated to produce 4000 seeds in 170 days or 90seeds in 80 days as compared to 200 seeds in 120 days under normalgrowing conditions represented by the control method (see Table 2). Themethods described herein solve a wide range of seed production needsrequired during research, regulatory, breeding, and commercial phases ofproduct development. For example, the amount of seeds produced and theamount of time required to produce such seeds can be varied depending onthe need in a particular product development phase.

In a particular application of the methods of the present invention,increasing the amount of seeds produced from a single plant can reducethe amount of work required for molecular characterization of pure seedsfor commercial production. For example, 12-18 sibling lines aretraditionally bulked to create a commercial seed lot with confirmedgenetic purity. However, using the methods described herein, Applicantshave demonstrated that only one or two plants are needed to produce acommercial seed lot, thus significantly reducing the amount of qualityassurance and/or quality control assays required on the sibling lines.

Increasing the amount of seeds produced from a single plant may also bebeneficial so that archiving, biochemical analyses e.g., oil and fattyacid analyses, and germination studies can be completed using seeds froma single plant thus reducing the variation in source material when morethan one plant is used.

Further benefits of the present invention can be achieved by producingmore developing pods from a single plant. More developing pods providesfor more immature embryos which in turn can supply the relatively largeamounts of protein normally required for conducting studies forregulatory dossier.

Producing more seeds from a single plant may further enables theidentification of more progeny seeds with an acceptable molecularprofile, e.g., seeds with single copy inserts without the vectorbackbone expressing a gene of interest at an efficacious level, fromplants transformed with 2T constructs or multiple-traits where theprobability of finding a desired seed is lower in a population of seeds.For example, only 1 out of 256 seeds is likely to contain a triplehomozygous marker-free plant. With a large number of seeds produced froma single plant, it is easier to identify such a seed.

The methods described herein may further reduce generation time whichcan enable rapid advancement of a seed to meet specific field plantingdeadlines, obtain acceptable molecular profiles and sufficient seedyields, and provide improved efficiency of large grow-outs due to highplant density.

Further, the methods of the present invention may eliminate the need forShort Day Greenhouses as these are difficult to cool in summer. Thewarm, dark conditions encountered in the summer during night in a ShortDay Greenhouse cause flower abortion and excessive night respirationstress. These Greenhouses are expensive to build and older greenhousemay be difficult to retrofit.

Without being held to a particular theory, experience to date suggeststhat the methods described herein appear to disrupt, enhance, or competewith a plant's normal circadian rhythm to trigger early flowering onphysiologically young plants. Thus, Applicants believe that the methodmay enhance yield by inducing indeterminate flowering, more branching,shorter internodes, and more flowers and pod set per internode.

The methods of the present invention allow a grower to customize theseed yield and generation time to meet specific business needs. The perplant yields can be increased by up to 30 fold (from 200 seeds to 6000seeds) or the generation time can be decreased by 30% (250 seeds in 90days vs. 120 days).

The methods can also be performed year round and could eliminate theneed for winter nurseries thus increasing the throughput of the productdevelopment process and reducing the total time needed to test aparticular seed for developing the commercial seed. Producing more seedsenables conducting field trials in a single location several monthsfaster than the seeds produced with the current Short Day methods.Current short day methods require an extra seed increase generation stepin the field to obtain sufficient seeds for conducting a field trial inone location in a subsequent year. The time savings of several monthscould lead to earlier commercial launch dates and ultimately additionalproduct revenues.

A method for manipulating yield of a short day plant is provided. Themethod comprises initiating growth of at least one short day plant underlong day growing conditions. The short plant is selected from the groupconsisting of soybean, cotton, rice, sugarcane, tobacco, and strawberry.In one embodiment, the long day growing conditions comprise at leastabout 14 hours of light per day at a light intensity of from about 1000to about 2000 μmoles m⁻¹s⁻¹ and a temperature of from about 84° F. toabout 90° F. and a night temperature of from about 62° F. to about 70°F. In another embodiment, the long day growing conditions comprise about18 hours of light per day at a light intensity of about 2000 μmoles m⁻¹s⁻¹ and a temperature of about 86° F. and a night temperature of about68° F.

The method further comprises controlling the environment of the shortday plant to provide for short day growing conditions for about 3 toabout 21 days. The period of short day growing conditions is initiatedat a plant growth stage of from about V1 to about V4. In one embodiment,the short day growing conditions comprise maintaining from about 9 toabout 11 hours of light per day at a light intensity of from about 700to about 900 μmoles μmoles m⁻¹ s⁻¹ and a temperature of from about 78°F. to about 82° F. and about 14 hours of night at a temperature of fromabout 66° F. to about 70° F. In another embodiment, the short daygrowing conditions comprise maintaining about 10 hours of light per dayat a light intensity of about 900 μmoles m⁻¹ s⁻¹ and a temperature ofabout 80° F. and about 14 hours of night at a temperature of about 68°F.

The method further comprises returning the plant to long day growingconditions. As described above, in one embodiment, the long day growingconditions comprise at least about 14 hours of light per day at a lightintensity of from about 1000 to about 2000 μmoles m⁻¹s⁻¹ and atemperature of from about 84° F. to about 90° F. and a night temperatureof from about 62° F. to about 70° F. In another embodiment, the long daygrowing conditions comprise about 18 hours of light per day at a lightintensity of about 2000 μmoles m⁻¹ s⁻¹ and a temperature of about 86° F.and a night temperature of about 68° F. In current green house methodsand field production methods plants are typically allowed to grow undershort day or under decreasing day light conditions until seeds areharvested. Without being held to a particular theory, Applicants believethat returning the plant to long day growing conditions may send avegetative signal to already reproductive plants, providing up to 800more hours of photosynthesis than the current greenhouse method andresulting in increased branching, shorter internodes, and more pods perinternode. Longer photoperiods also allow the night temperature to bereduced, which results in very high self-pollination rates and pod setcompared to the current greenhouse method where higher nighttemperatures can lead to a higher rate of flower abortion and lower podset. In addition to providing a constant long day photoperiod, theinstant methods may include a gradually increased long day photoperiodfrom about 16 hours to about 20 hours over a period of 3 weeks.Gradually increasing long day photoperiods may send an additional longday signal to the plant thereby creating plants with very high averageseed yields of about 2000 to about 4000 seeds.

In alternative embodiments, the methods of the present invention mayfurther comprise growing the short day plant under conditions thatrestrict vegetative growth and enhance flowering. Such conditions mayinclude growing the short day plant in a soil volume of from about 2.0mL to about 4.0 mL for every seed to be produced. This can be achievedby controlling pot size. Different pot sizes can be used to increase ordecrease the flowering response. Generally, smaller pot size will reducevegetative growth and increase the flowering response leading to verydeterminate growth habit and larger pot size will increase vegetativegrowth and allow for indeterminate flowering, more pods, and more seeds.

In still other embodiments, the methods of the present invention mayfurther comprise providing the short day plant with nutrients sufficientto support seed development. In one embodiment, such nutrients can beselected from the group consisting of Calcium Nitrate, Phosphate,micronutrients, and Magnesium Sulfate. In another embodiment, thenutrients are supplied in amounts sufficient to provide a soil EC ofabout 1.0 to about 1.6 mmhos and a soil pH of from about 5.1 to about6.0. The nutrients can be provided by utilizing advanced irrigationtechniques such as soil-less media, continuous liquid fertilization, andoptimal moisture management. Applicants have discovered that under theseconditions, the plants become root-bound, contributing to the vegetativeand flowering signals needed for enhanced yield. Under root-boundconditions, the plants still require complete mineral nutrition andmoisture. This is achieved by administering fertilizer solutions severaltimes to each pot.

A method for manipulating yield of a soybean plant is also provided. Themethod comprises initiating growth of at least one soybean plant underlong day growing conditions. In one embodiment, the long day growingconditions comprise at least about 14 hours of light per day at a lightintensity of from about 1000 to about 2000 μmoles m⁻¹s⁻¹ and atemperature of from about 84° F. to 90° F. and a night temperature offrom about 62° F. to about 70° F. In another embodiment, the long daygrowing conditions comprise about 18 hours of light per day at a lightintensity of about 2000 μmoles m⁻¹ s⁻¹ and a temperature of about 86° F.and a night temperature of about 68° F.

The method further comprises controlling the environment of the soybeanplant to provide for short day growing conditions for about 3 to about21 days. The period of short day growing conditions is initiated at aplant growth stage of from about V1 to about V4. In one embodiment, theshort day growing conditions comprise maintaining about 9 to about 11hours of light per day at a light intensity of from about 700 to about900 μmoles μmoles m⁻¹ s⁻¹ and a temperature of from about 78° F. toabout 82° F. and about 14 hours of night at a temperature of from about66° F. to about 70° F. In another embodiment, the short day growingconditions comprise maintaining about 10 hours of light per day at alight intensity of about 900 μmoles m⁻¹ s⁻¹ and a temperature of about80° F. and 14 hours of night at a temperature of about 68° F.

The method further comprises returning the plant to long day growingconditions as described above.

In another embodiment, the method of the present invention furthercomprises growing the soybean plant under conditions that restrictvegetative growth and enhance flowering. Such conditions may includegrowing the soybean plant in a soil volume of from about 2.0 mL to about4.0 mL for every seed to be produced. This can be achieved bycontrolling pot size. Different pot sizes can be used to increase ordecrease the flowering response. Generally, smaller pot size will reducevegetative growth and increase the flowering response leading to verydeterminate growth habit and larger pot size will increase vegetativegrowth and allow for indeterminate flowering, more pods, and more seeds.

In yet another embodiment, the method of the present invention furthercomprises providing the soybean plant with nutrients sufficient tosupport seed development. In one embodiment, such nutrients can beselected from the group consisting of Calcium Nitrate, Phosphate,micronutrients, and Magnesium Sulfate. In another embodiment, thenutrients are supplied in an amount sufficient to provide a soil EC ofabout 1.0 to about 1.6 mmhos and a soil pH of about 5.1 to about 6.0.The nutrients can be provided by utilizing advanced irrigationtechniques such as soil-less media, continuous liquid fertilization, andoptimal moisture management. Applicants have discovered that under theseconditions, the plants become root-bound, contributing to the vegetativeand flowering signals needed for enhanced yield. Under root-boundconditions, the plants still require complete mineral nutrition andmoisture. This is achieved by administering fertilizer solutions severaltimes to each pot.

A method for identifying yield genes from a short day plant is provided.The method comprises: initiating growth of at least one short day plantunder long day growing conditions; controlling the environment of the atleast one short day plant to provide for short day growing conditionsfor about 3 to about 21 days; returning the plant to long day growingconditions; and performing transcriptional profiling from a tissueharvested from the plant grown in step b) and c) to identify yieldgenes.

The short day plant is selected from the group consisting of soybean,cotton, rice, sugarcane, tobacco, and strawberry. In one embodiment theshort day plant is a soybean plant.

The long day growing conditions comprise at least about 14 hours oflight per day at a light intensity of from about 1000 to about 2000μmoles m⁻¹s⁻¹ and a temperature of from about 84° F. to about 90° F. anda night temperature of from about 62° F. to about 70° F.

The method further comprises the step of controlling the environment ofthe short day plant to provide for short day growing conditions forabout 3 to about 21 days. The period of short day growing conditions isinitiated at a plant growth stage of from about V1 to about V4. Theshort day growing conditions comprise maintaining about 9 to about 11hours of light per day at light intensity of about 700 to about 900μmoles μmoles m⁻¹ s⁻¹ and a temperature of from about 78° F. to about82° F. and about 14 hours of night at a temperature of from about 66° F.to about 70° F.

The method further comprises returning the plant to long day growingconditions as described above.

The method further comprise the step of performing transcriptionalprofiling from a tissue harvested from the plant grown in step b) and c)to identify yield genes. The yield genes comprise genes that areinvolved in induction of early flowering, pod set, retention of flowersand pods, and abscission of flowers and pods.

Various cultivars of soybean can be used cultivar for identifying yieldgenes. Plants are first grown under long day growing conditions asdescribed above until the plants reach V2-V3 stage. Then plants aretransferred to short day growing conditions as described above. Plantsare sampled at one, three and five days after the experimental plantsare transferred to short day growing conditions. Fully-expanded leavesas source leaves and shoot apices are suitable tissue for identifyingdifferentially expressed genes under experimental and controlconditions. The tissue sampling is done at the V3 stage. The samples areimmediately frozen in liquid nitrogen and stored at −80° C. prior to RNAextraction for transcription profiling.

For identifying genes that are involved in retention of flowers andpods, and/or abscission of flowers and pods, soybean plants were grownas above. After flowering and right before sampling, half the plants aretreated with short day conditions to facilitate abscission of theirflowers and pods. Plants are sampled at two, four and six days after thetransfer to short day conditions and nine hours after light come on.Fully-expanded leaves from a side branch as source leaves and top leavesof the branch as sink leaves and newly opened flower buds prior to andpost pollination are suitable tissues for identifying differentiallyexpressed genes under experimental and control conditions. The samplesare immediately frozen in liquid nitrogen and stored at −80° C. prior toRNA extraction for transcription profiling.

Several means can be utilized to identify differentially expressed genesin experimental and control plants and are well known to those skilledin the art. These include serial analysis of gene expression (SAGE,SuperSAGE) and gene expression profiling. In one aspect of theinvention, gene expression or transcriptional profiling is used toidentify yield genes by comparing their differential expression underexperimental and control conditions.

RNA is extracted from the pooled samples using a pre-manufactured kitand protocol by OmegaBiotek. A custom made soybean genome expressionmicroarray chip from Affymetrix is used. The microarray contains 1.4million features (each 11 micron in size) covering 83 thousand genes andsome negative alien sequences per array. RNA is checked for quality byestimating OD at 260/280 ratio using nanodrop8000 and quality of 28s/18sribosome bands using Agilent Bioanalyzer2000. Three hundred nanogram RNAper sample was used in RT/IVT amplification and labeling procedure asprovided by InVitrogen and Epicentre. Labeled cRNA probe is fragmentedand hybridized to the array. The hybridization, washing, detection, andscanning are done according to Affymetrix protocol.

Analysis is done by Robust Multi Array (RMA) algorithms to performbackground correction, global normalization and summarization ofintensity data adjusted using the 75^(th) percentile. The intensity datais converted to log base2 prior to the statistical analysis, which usesANOVA models to analyze the data set and perform the comparisons betweendata set from experimental and control samples. Differentially expressedgenes are identified by using a threshold with a false discovery rate of5%, a raw probability coefficient of 0.0001 and a 1.5 fold change orgreater as the standard for significance. FunCat analysis is used toidentify over-representation of functional categories based onmolecular, biochemical and cellular characteristics of the proteinsencoded by the transcripts. K-means cluster analysis is used to groupgenes based on similarities of expression profiles, systems networkbuilding, and promoter motif analysis.

In another embodiment, the method of the present invention furthercomprises growing the short day plant under conditions that restrictsvegetative growth and enhances flowering. Such conditions comprisegrowing the short day plant in a soil volume of about 2.0 mL to about4.0 mL per seed to be produced.

In another embodiment, the method of the present invention furthercomprises providing the short day plant nutrients sufficient to supportseed development. Such nutrients may be selected from the groupconsisting of Calcium Nitrate, Phosphate, micronutrients, and MagnesiumSulfate wherein the amount of nutrients supplied provides a soil EC offrom about 1.0 to about 1.6 mmhos and a soil pH of from about 5.1 toabout 6.0.

EXAMPLES Example 1

This example describes a method for manipulating vegetative andflowering responses in soybean, a short day plant, with external signalsfor decreasing or increasing seed yield and manipulating seed generationtime.

Soybean seeds were sown as one seed per 200 mL pot (McConkey Company,Sumner, Wash.) loosely filled with the Sunshine LP5 soil (Sun GroHorticulture, Vancouver, BC, Canada) and allowed to germinate and growunder long day conditions in a Green House (GH) or a growth chamber.

The final potting soil was prepared by mixing 3.8 cu.ft. bales ofSunshine #1 soil (Sun Gro Horticulture, Vancouver, BC, Canada) manuallyor in Gleason batch soil mixer (Hummert International, Earth City, Mo.,USA). Eighty mL of APEX® micronutrients and 1000 mL of APEX® 14-14-14(J.R. Simplot Company, Lathrop, Calif., USA) controlled releasefertilizer was added to each bale of soil. Mixed soil was transferred todesired pot sizes. A Saturated Media Extraction test was performed onthe mixed soil. The pots filled with mixed soil were watered withReverse Osmosis water to saturate the soil and until water started toleach. Electro-Conductivity (EC) and pH measurements were taken on theleached water using an EC and pH meter (MYRON L COMPANY, Carlsbad,Calif., USA) such that the EC was in the range of 3.5 to 7 mS and pH wasin the range of 5.2-5.7. If the EC was higher than 7, then soil wascontinually flushed with Reverse Osmosis water until the EC was below 7.

The long day growing conditions were as follows. A photoperiod of 16-18hours was provided using supplemental lighting to accumulate between40-60 moles of total light per day. The temperatures were set based onthe season and the weather to ensure target temperatures such that toaccumulate as many hours per day at or above 86° F. to maximizephotosynthesis and minimize night respiration with cooler temperatures.The target temperatures for the cool season were: day, 86° F.-90° F. andnight, 68° F.-70° F. and the warm season were: day, 84° F.-88° F. andnight, 66° F.-68° F. The ambient CO₂ and humidity was maintained below65%.

A constant and low concentration of nutrient solution and optimalmoisture content was also provided to the plants. The plants wereirrigated from 1 to 6 times a day depending upon the climate and the potsize. The pots were fertilized with a nutrient solution having acomposition and characteristics shown in Table 1.

The short day growing conditions were applied in a growth chamber (GC)e.g., PGR15, PGC20 or GR144 (Conviron, Controlled Environments Inc.,Pembina, N. Dak., USA) as follows. Soybean plants at a suitable V stage(e.g., V1 to V4; see Table 2, column 2) were transferred to the GC.Briefly, V1 stage is when first set of tri foliate leaves are unfolded,V2 stage is when the first trifoliate leaf is fully expanded, and V3 iswhen the second trifoliate leaf is fully expanded. A V-stage is a goodmeasure of physiological stage but vigor must also be used to determinethe correct stage. A V3 plant with low vigor may be equivalent to a V2plant with good vigor. Plants seeded on the same day usually havedifferent development rates. For example, A3525 control take 11 daysafter seeding to reach V2 and some R1 transgenic seeds may take 21 daysto reach V2 stage. The plants were grown at a photoperiod of 10 hours atlight intensity of 700-900 μmoles/m/s and temperature of 78-82° F. and14 hours of night at a temperature of 66-68° F. The plants wereirrigated with a nutrient solution (Table 1) in order to provide optimalgrowth conditions depending upon the size of the plant and the weatherconditions. Each plant was grown at a plant density of 0.18 sq. ft. inthe GC. The plants were kept in the GC from about 3 to 21 days dependingupon the yield needed (see Table 2, column 3).

After subjecting the plants to short day growing conditions, the plantswere returned to the long day growing conditions provided in the greenhouse (GH) as described above. The plants were first transplanted tolarger pots depending upon the yield needed (Table 2, column 4). Thepots were prepared as described above. Maximum pot densities in the GHwere dependent upon the pot size and were as follows: 200 mL pot/0.5 sq.ft.; 750 mL pot/1 sq. ft.; 2.7 L pot/2 sq. ft.; and 8.6 L pot/4 sq. ft.

As shown in Table 2, whereas the current short day method yielded only200 seeds in 120 days, the method of present invention yielded anywherefrom 90 seeds to 4000 seeds in 80 to 170 days depending upon the shortday induction stage, short day induction period, pot size, and long dayperiod after flowering.

TABLE 1 Composition and characteristics of the nutrient solution used inthe present invention. Macro (NPK) and Micro (Rest) nutrients are inppm. pH EC Alkalinity 5.2-5.6 1.2-1.6 3.86 Ca Mg Na 124 48.2 2.93 Cl BFe Mn 3.05 0.326 1.44 0.136 Cu Zn Mo Al 0.036 0.099 0.024 0.079 NO3-NNH4-N Total N 104 12.1 116 S P K 102 26.5 131

TABLE 2 Manipulation of seed yield in soybean using the method of thepresent invention. SD Induction Generation Pot Induction period Pot SeedTime Density/ Seeds/ Induction Name Stage (days) Size Yield (days) sq ftSq ft 1 2 3 4 5 6 7 8 1 Super Rapid Cycle V1 21 300 mL 90 80 0.67 134 2Rapid Cycle V2 14 750 mL 250 90 1 250 3 Regular Induction V2-4 14 2.7 L500 120 2 250 4 Super Plant V2-4 7 8 L 2000 150 5 400 5 Super Plant PlusV2-4 7 10 L 4000 170 8 500 6 Current Short Day V6 80 8 L 200 120 1 200Method

Example 2

This example demonstrates extension of the methods described herein tocotton plants.

Short day cotton has an indeterminate vegetative growth habit under longday growth conditions and normally produces very little seed with highamounts of vegetative growth. However, Applicants believe that a highseed yielding cotton plant can be produced by applying the methods ofthe present invention to a short day cotton variety. Cotton seeds willbe germinated under long day conditions and seedlings will be subjectedto short day induction conditions for 5 and 10 days at the first andthird unifoliate stages to trigger early flowering. Seedlings will thenbe subjected to long day conditions. The same variations of experimentalconditions as described in Example 1 including long day growthconditions, short day induction conditions, light intensity, soil types,temperature, pot size and nutrients will be applied on the cotton plantsto produce a high yielding phenotype.

Example 3

This example demonstrates a method of the invention usingtranscriptional profiling to identifying potential genes conferringincreased yield in short day plants.

To identify genes involved in floral initiation, pod initiation, flowerset, and pod set, soybean plants of cultivar A3555 are grown in 200 mlpots under long day growing conditions (17 hours of daylight) until theplants reach V2-V3 stage and all the plants are transplanted into 4 Lpots. Half the plants are then transferred to short day conditions (10hours of daylight) for 7 days at 26° C. during the day, 19° C. at nightat a light intensity of about 800 μE. Control plants continue to growunder long day growth conditions. Plants are sampled at one, three andfive days after the experimental plants are transferred to short dayconditions. Plants are sampled one hour and nine hours after lights comeon. Fully-expanded second trifoliate leaves on the fourth node arecollected as source leaves and pooled. For apex tissue, meristem tissueand non-expanded primordial leaf are collected and pooled. The tissuesampling is done at the V3 stage. The samples are immediately frozen inliquid nitrogen and stored at −80° C. prior to RNA extraction fortranscription profiling.

To identify genes that are involved in retention of flowers and pods,and/or abscission of flowers and pods, soybean plants of cultivar A3555are grown in 200 ml pots under long day growing conditions (17 hours ofdaylight) until the plants reach V2-V3 stage. Plants are thentransplanted into 4 L pots grown under short day conditions (10 hours ofdaylight) for 7 days at 26° C. during the day at a light intensity ofabout 800 μE, and at 19° C. at night. After short day conditions, plantsare transferred to long day conditions. After flowering and right beforesampling, half the plants are treated with short day conditions tofacilitate abscission of their flowers and pods. Plants are sampled attwo, four and six days after the transfer to short day conditions andnine hours after light come on. Fully-expanded leaves from a side branchbetween the fourth node and internode are collected as source leaves andpooled whereas top leaves of the branch are collected as sink leaves andpooled. Newly opened flower buds two days prior to and one day postpollination are collected from the entire plant and pooled. The samplesare immediately frozen in liquid nitrogen and stored at −80° C. prior toRNA extraction for transcription profiling.

RNA is extracted from the pooled samples using a pre-manufactured kitand protocol by OmegaBiotek. A custom made soybean genome expressionmicroarray chip from Affymetrix is used. The microarray contains 1.4million features (each 11 micron in size) covering 83 thousand genes andsome negative alien sequences per array. RNA is checked for quality byestimating OD at 260/280 ratio using nanodrop8000 and quality of 28s/18sribosome bands using Agilent Bioanalyzer2000. Three hundred nanogram RNAper sample was used in RT/IVT amplification and labeling procedure asprovided by InVitrogen and Epicentre. Labeled cRNA probe is fragmentedand hybridized to the array. The hybridization, washing, detection, andscanning are done according to Affymetrix protocol.

Analysis is done by Robust Multi Array (RMA) algorithms to performbackground correction, global normalization and summarization ofintensity data adjusted using the 75^(th) percentile. The intensity datais converted to log base2 prior to the statistical analysis, which usesANOVA models to analyze the data set and perform the comparisons betweendata set from experimental and control samples. Differentially expressedgenes are identified by using a threshold with a false discovery rate of5%, a raw probability coefficient of 0.0001 and a 1.5 fold change orgreater as the standard for significance. FunCat analysis is used toidentify over-representation of functional categories based onmolecular, biochemical and cellular characteristics of the proteinsencoded by the transcripts. K-means cluster analysis is used to groupgenes based on similarities of expression profiles, systems networkbuilding, and promoter motif analysis.

All of the materials and methods disclosed and claimed herein can bemade and used without undue experimentation as instructed by the abovedisclosure. Although the materials and methods of this invention havebeen described in terms of preferred embodiments and illustrativeexamples, it will be apparent to those of skill in the art thatvariations can be applied to the materials and methods described hereinwithout departing from the concept, spirit and scope of the invention.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

1. A method for manipulating yield of a short day plant, the methodcomprising: initiating growth of at least one short day plant under longday growing conditions; controlling the environment of the at least oneshort day plant to provide for short day growing conditions for about 3to about 21 days; and returning the plant to long day growingconditions.
 2. The method of claim 1, wherein the at least one shortplant is selected from the group consisting of soybean, cotton, rice,sugarcane, tobacco, and strawberry.
 3. The method of claim 1, whereinthe period of short day growing conditions is initiated at a plantgrowth stage of from about V1 to about V4.
 4. The method of claim 1,wherein the long day growing conditions comprise at least about 14 hoursof light per day at a light intensity of from about 1000 to about 2000μmoles m⁻¹ s⁻¹ and a temperature of from about 84° F. to about 90° F.and a night temperature of from about 62° F. to about 70° F.
 5. Themethod of claim 1, wherein the step of controlling the environment ofthe at least one short day plant to provide for the short day growingconditions comprises maintaining about 9 to about 11 hours of light perday at a light intensity of from about 700 to about 900 μmoles μmolesm⁻¹ s⁻¹ and a temperature of from about 78° F. to about 82° F. and about14 hours of night at a temperature of from about 66° F. to about 70° F.6. The method of claim 1, further comprising growing at least one shortday plant under conditions that restrict vegetative growth and enhanceflowering.
 7. The method of claim 6, wherein the method furthercomprises growing the at least one short day plant in a soil volume offrom about 2.0 mL to about 4.0 mL of soil volume for every seed to beproduced.
 8. The method of claim 1, further comprising providing the atone short day plant nutrients sufficient to support seed development. 9.The method of claim 8, wherein the nutrients are selected from the groupconsisting of Calcium Nitrate, Phosphate, micronutrients, and MagnesiumSulfate and the amount of nutrients supplied provide a soil EC of about1.0 to about 1.6 mmhos and a soil pH of about 5.1 to about 6.0.
 10. Amethod for manipulating yield of a soybean plant, the method comprising:initiating growth of at least one soybean plant under long day growingconditions; controlling the environment of the at least one soybeanplant to provide for short day growing conditions for about 3 to about21 days; and returning the plant to long day growing conditions.
 11. Themethod of claim 10, wherein the at least one soybean plant is subjectedto short day growing conditions at a growth stage of about V1 to aboutV4.
 12. The method of claim 10, wherein the long day growing conditionscomprise at least about 14 hours of light per day at a light intensityof from about 1000 to about 2000 μmoles m⁻¹ s⁻¹ and a temperature offrom about 84° F. to about 88° F. and a night temperature of from about62° F. to about 68° F.
 13. The method of claim 10, wherein the step ofcontrolling the environment of the at least one short day plant toprovide for the short growing conditions comprises maintaining about 9to about 11 hours of light per day at a light intensity of from about700 to about 900 μmoles m⁻¹ s⁻¹ and a temperature of from about 78° F.to about 82° F. and about 14 hours of night at a temperature of fromabout 66° F. to about 70° F.
 14. The method of claim 1, furthercomprising growing the at least one soybean plant under conditions thatrestrict vegetative growth and enhance flowering.
 15. The method ofclaim 6, wherein the method further comprises growing the at least onesoybean plant in a soil volume of from about 2.0 mL to about 4.0 mL ofsoil volume for every seed to be produced.
 16. The method of claim 1,further comprising providing the at one soybean plant nutrients forsupporting seed development.
 17. The method of claim 8, wherein thenutrients are selected from the group consisting of Calcium Nitrate,Phosphate, micronutrients, and Magnesium Sulfate and the nutrients aresupplied in amounts sufficient to provide a soil EC of from about 1.0 toabout 1.6 mmhos and a soil pH of about 5.1 to about 6.0.
 18. A methodfor identifying genes conferring increased yield in a short day plant,the method comprising: initiating growth of at least one short day plantunder long day growing conditions; controlling the environment of the atleast one short day plant to provide for short day growing conditionsfor about 3 to about 21 days; returning the plant to long day growingconditions; harvesting tissue from the at least one short day plant; andperforming transcriptional profiling on the harvested tissue to identifygenes that may confer increased yield in the at least one short dayplant.
 19. The method of claim 18, wherein the at least one short plantis selected from the group consisting of soybean, cotton, rice,sugarcane, tobacco, and strawberry.
 20. The method of claim 18, whereinthe period of short day growing conditions is initiated at a plantgrowth stage of from about V1 to about V4.
 21. The method of claim 18,wherein the long day growing conditions comprise at least about 14 hoursof light per day at a light intensity of from about 1000 to about 2000μmoles m⁻¹s⁻¹ and a temperature of from about 84° F. to about 90° F. anda night temperature of from about 62° F. to about 70° F.
 22. The methodof claim 18, wherein the step of controlling the environment of the atleast one short day plant to provide for the short day growingconditions comprises maintaining about 9 to about 11 hours of light perday at a light intensity of from about 700 to about 900 μmoles m⁻¹s⁻¹and a temperature of from about 78° F. to about 82° F. and about 14hours of night at a temperature of from about 66° F. to about 70° F. 23.The method of claim 18, further comprising growing at least one shortday plant under conditions that restrict vegetative growth and enhanceflowering.
 24. The method of claim 18, wherein the method furthercomprises growing the at least one short day plant in a soil volume offrom about 2.0 mL to about 4.0 mL of soil volume for every seed to beproduced.
 25. The method of claim 18, further comprising providing theat one short day plant nutrients sufficient to support seed development.26. The method of claim 25, wherein the nutrients are selected from thegroup consisting of Calcium Nitrate, Phosphate, micronutrients, andMagnesium Sulfate and the amount of nutrients supplied provide a soil ECof about 1.0 to about 1.6 mmhos and a soil pH of about 5.1 to about 6.0.27. The method of claim 18, wherein the genes conferring increased yieldon the at least one short day plant comprise genes inducing earlyflowering, pod set, retention of flowers and pods, or abscission offlowers and pods.